In search for novel antiseizure drugs (ASDs), the European FP7-funded PharmaSea project used zebrafish embryos and larvae as a drug discovery platform to screen marine natural products to identify promising antiseizure hits in vivo for further development. Within the framework of this project, seven known heterospirocyclic γ-lactams, namely, pseurotin A, pseurotin A, pseurotin F1, 11- O-methylpseurotin A, pseurotin D, azaspirofuran A, and azaspirofuran B, were isolated from the bioactive marine fungus Aspergillus fumigatus, and their antiseizure activity was evaluated in the larval zebrafish pentylenetetrazole (PTZ) seizure model. Pseurotin A and azaspirofuran A were identified as antiseizure hits, while their close chemical analogues were inactive. Besides, electrophysiological analysis from the zebrafish midbrain demonstrated that pseurotin A and azaspirofuran A also ameliorate PTZ-induced epileptiform discharges. Next, to determine whether these findings translate to mammalians, both compounds were analyzed in the mouse 6 Hz (44 mA) psychomotor seizure model. They lowered the seizure duration dose-dependently, thereby confirming their antiseizure properties and suggesting activity against drug-resistant seizures. Finally, in a thorough ADMET assessment, pseurotin A and azaspirofuran A were found to be drug-like. Based on the prominent antiseizure activity in both species and the drug-likeness, we propose pseurotin A and azaspirofuran A as lead compounds that are worth further investigation for the treatment of epileptic seizures. This study not only provides the first evidence of antiseizure activity of pseurotins and azaspirofurans, but also demonstrates the value of the zebrafish model in (marine) natural product drug discovery in general, and for ASD discovery in particular.

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In order to accelerate the isolation and characterisation of structurally new or novel secondary metabolites, it is crucial to develop efficient strategies that prioritise samples with greatest promise early in the workflow so that resources can be utilised in a more efficient and costeffective manner. We have developed a metrics-based prioritisation approach using exact LC-HRMS which uses data for 24,618 marine natural products held in the PharmaSea database. Each sample was evaluated and allocated a metric score by a software algorithm based on the ratio of new masses over the total (sample novelty), ratio of known masses over the total (chemical novelty), number of peaks above a defined peak area threshold (sample complexity), and peak area (sample diversity). Samples were then ranked and prioritized based on these metric scores. To validate the approach, 8 marine sponges and 6 tunicate samples collected from the Fiji Islands were analysed, metric scores calculated and samples targeted for isolation and characterisation of new compounds. Structures of new compounds were elucidated by spectroscopic techniques, including 1D and 2D NMR, MS and MS/MS. Structures were confirmed by Computer Assisted Structure Elucidation methods (CASE) using the ACD/Structure Elucidator Suite.

A secondary ion mass spectrometer (SIMS) instrument is described that is configured with two SIMSdetectors that are both low-field extraction, quadrupole-based filters. Secondary ions are generated by sputtering with a liquid-metal ion gallium source and column of the type that is common on two-beam electron microscopes. The gallium ion beam, or focused ion beam achieves sub-100 nm focus with a continuous current of up to 300 pA. Positive secondary ions are detected by one SIMSdetector, and simultaneously, negative secondary ions are detected by the second SIMSdetector. The SIMSdetectors are independently controlled for recording mass spectra, concentration depth profiles, and secondary ion images. Examples of simultaneous positive and negative SIMS are included that demonstrate the advantage of this facility for surface analysis and depth profiling. The SIMS secondary ion collection has been modeled using the ray tracing program simion (“simion”, Scientific Instrument Services, Inc., Ringoes, NJ, 08551-1054, see http://www.simion.com) in order to understand the interaction of the secondary ions of opposite polarities in the extraction volume for the purpose of optimizing secondary ion collection

Two microscopes with focused ion beam (FIB) columns in the Department of Materials, Imperial College, have been fitted with SIMS probes (Hiden EQS 1000). Both probes have differential pumping to maintain the vacuum in the probe below 5 × 10 À6 mbar irrespective of the main operating and imaging conditions of the microscopes. In both microscopes, standard operations include the injection of oxygen-containing species close to the sputter site. This process is also a technique for SIMS analysis instrumentation to enhance positive ion yields during sputtering where an oxygen primary ion beam for dynamic SIMS analysis is not available. The aim of this paper is to describe the compatibility of the differentially pumped SIMS probe with the normal specifications of microscopes. In particular, the modern field emission gun-SEM instrument's (Zeiss Auriga) ability to provide ultra-high spatial resolution SIMS in combination with electron microscopy imaging, energy dispersive spectroscopy and electron backscatter diffraction. In the second instrument, an ion microscope (FEI FIB200), the installed system is additional to the existing FEI SIMS probe. Simultaneous detection of positive and negative secondary ions from the same sputtered crater is obtained on this instrument with the two SIMS probes. Figure 3. Crater for SIMS depth profile in FEI FIB200 after depth profile. The inset is an image of the crater wall obtained by first tilting the silicon surface by 45°. R. J. Chater et al. wileyonlinelibrary.com/journal/sia